The exocrine pancreas is responsible for the synthesis and secretion of digestive enzymes into the intestine. The acinar cell is responsible for the pancreas' exocrine functions and can be characterized as a polarized secretory epithelia. Digestive enzyme secretion is also regulated and can be stimulated with acetylcholine and cholecystokinin. The key subcellular organelle responsible for regulated secretion in the acinar cell is the zymogen granule; a secretory vesicle that stores and concentrates digestive enzymes until secretion is stimulated. The focus of this project has been the characterization of zymogen granule membrane proteins as a means toward understanding the mechanisms underlying the formation of secretory granules and the targeting of proteins to the regulated secretory pathway. GP2 is the dominant protein in the zymogen granule membrane and accounts for 35 percent of the total granule membrane protein. In vitro studies have demonstrated that GP2 is able to aggregate with other exocrine regulated secretory proteins in acidic conditions designed to mimic the trans-Golgi network and immature secretory granule where sorting occurs. GP2 is initially bound to the membrane through a glycosylphosphotidylinositol linkage, which by itself confers membrane protein sorting to the apical plasma membrane. Because GP2 exhibits binding to the soluble digestive enzymes within the granule and contains a sorting determinant for the apical plasma membrane, it is likely that the protein plays a significant role in sorting digestive enzymes into the zymogen granule and the regulated pathway. The goal of this application for the next funding period is to define GP2's function. Transgenic knockout techniques will be employed to produce a mouse with a GP2 null allele. Because GP2 is specifically expressed in the pancreatic zymogen granule and the exocrine pancreas is not functional until after birth, it is unlikely that an embryonic lethal will result from the mutation. Thus preparations have been made to analyze the resultant mutant mice using biochemical, morphological, and physiological approaches. Electron microscopy will be used to study GP2's role on the formation of the zymogen granule. Primary pancreatic cultures will be used to study the integrity of the regulated secretory pathway in the mutants. To establish that any resultant phenotypes are truly secondary to the GP2 null mutant, preparations have been made for the reconstitution of wild-type GP2 in primary pancreatic cultures using adenovirus mediated gene delivery. Adenovirus expression of a variety of mutant GP2 constructs will be used to identify important functional domains in the protein. Last, studies will be performed on the effects of the GP2 mutation in experimentally induced pancreatitis. The model we propose to generate will provide important information on GP2 biology and may also provide potential models for human acute and chronic pancreatic diseases.